Plasma membrane intramembranous particle topography in 3T3 and SV3T3 cells: the effect of cytochalasin B (original) (raw)

Influence of cell cycle and cell movement on the distribution of intramembranous particles in contact-inhibited and transformed cells

Experimental Cell Research, 1974

Freeze fracture ultrastructure studies have shown that contact inhibited 3T3 cells contain aggregated intramembranous particles (IMP) while transformed 3T3 cells have randomly distributed IMP. The results of this study show that the aggregation of IMP in 3T3 cells is primarily related to the degree of cell contact and not significantly affected by inhibition of cell movement. Cell cycle studies do, however, show a transient disaggregation of IMP during the mitotic phase of the cell cycle. These observations are interpreted to suggest that changes in membrane structure which occur during mitosis or following cell-to-cell contact may be associated with changes in membrane fluidity and the activity of membrane enzymes that appear to be critical for control of cell growth and cell division.

Changes in Membrane Structure Associated with Cell Contact

Proceedings of the National Academy of Sciences, 1973

Ultrastructural analysis of 3T3 fibroblasts by freeze-cleavage has demonstrated significant changes in cell-membrane structure associated with cellto-cell contact and malignant transformation. These changes consist of a rearrangement and redistribution of intramembranous particles on the membrane fracture faces exposed by freeze-cleavage. The results show that noncontacted 3T3 cells in low density contain randomly distributed intramembranous particles. With the development of cell-to-cell contacts during the logarithmic phase of growth however, a pronounced aggregation of intramembranous particles is seen. A direct correlation

Differences in Membrane Fluidity and Structure in Contact-Inhibited and Transformed Cells

Proceedings of the National Academy of Sciences, 1974

Studies on contact-inhibited mouse embryo fibroblast 3T3 cells and 3T3 cells transformed by oncogenic RNA and DNA viruses and by a chemical carcinogen have demonstrated differences in plasma membrane architecture. Spin-label and freeze-fracture ultrastructural studies have shown that contact-inhibited cells have ordered membrane lipids and aggregated intramembranous particles, whereas transformed cells have fluid membrane lipids and randomly distributed intramembranous particles. These findings suggest a model for how changes in the cell membrane may account for some of the characteristic differences observed between contact-inhibited and transformed cells.

Plasma membrane alteration associated with malignant transformation in culture

Proceedings of the National Academy of Sciences, 1975

The intramembrane organization of the plasma membranes of nonmalignant cells in culture has been compared by freeze-fracturing with that of virally-transformed malignant cells. No dramatic differences are present in the distribution of intramembrane particles in the plasma membranes of these cells when the cells are examined without fixation or with mild fixation (glutaraldehyde treatment) prior to freezing. However, a redistribution of intramembrane particles into aggregates occurs in the membranes of nontransformed cells after treatment with glycerol. The

Dynamic study of intramembranous particles in human fresh erythrocytes using an “in vitro cryotechnique”

Microscopy Research and Technique, 2006

For analyses of dynamic ultrastructures of erythrocyte intramembranous particles (IMPs) in situ, a quick-freezing method was used to stabilize the flow behavior of erythrocytes embedded in vitreous ice. Fresh human blood was jetted at various pressures through artificial tubes, in which the flowing erythrocytes were elongated from biconcave discoid shapes to elliptical ones, and quickly frozen in liquid isopentane-propane cryogen (À1938C). They were freeze-fractured using a scalpel in liquid nitrogen, and routinely prepared for replica membranes. Many IMPs were observed on the protoplasmic freeze-fracture face (P-face) of the erythrocyte membranes. Some control erythrocytes under nonflowing or stationary conditions showed IMPs with their random distribution. However, other jetted erythrocytes under flowing conditions showed variously sized IMPs with much closer distribution. They were also arranged into parallel rows in some parts, and aggregated together. This quick-freezing method enabled for the first time the visualization of time-dependent topology and the molecular alteration of IMPs in dynamically flowing erythrocytes. Microsc. Res. Tech. 69: 291-295,

Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells

Experimental Cell Research, 1975

Differences in the distribution of plasma membrane intramembranous particles (PMP) have been demonstrated in normal and transformed fibroblasts using freeze fracture and electron microscopy. Transformed 3T3 cells contain randomly distributed PMP and contact-inhibited 3T3 cells have aggregated PMP when frozen in medium, glycerol, sucrose, or following stabilization in I % formaldehyde. To define some of the mechanisms controlling the organization of PMP in this system we have examined the effects of microtubule disruptive drugs including vinblastine sulfate and colchicine on SV3T3 cells. These drugs were observed to induce a dose-and time-dependent aggregation of PMP at concentrations between 10m9 and 10W5 M. These results suggest that modulation of PMP distribution in these cells may be influenced by an interaction of microtubules with plasma membrane components. However, the observation that lumicolchicine, a derivative of colchicine which does not disrupt microtubules, also promotes PMP aggregation, suggests that these drugs may also have a primary effect on the plasma membrane in addition to the disruption of microtubules. This is supported by the observation that reduced temperature (4°C) which is known to disrupt microtubules fails to induce PMP aggregation in SV3T3 cells, suggesting the hypothesis that changes in the interaction of plasma membrane or plasma membrane associated constituents may control the distribution of PMP in this cell system.

Changes in Intramembranous Particle Topography and Concanavalin A Receptor Mobility Associated with Myoblast Differentiation

Differentiation, 1979

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fured with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfiied live cells are reacted at 37O C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.

Hindered Diffusion of Inert Tracer Particles in the Cytoplasm of Mouse 3T3 Cells

Proceedings of The National Academy of Sciences, 1987

Using fluorescence recovery after photobleaching, we have studied the diffusion of fluorescein-labeled, size fractionated Ficoll in the cytoplasmic space of living Swiss 3T3 cells as a probe of the physical chemical properties of cytoplasm. The results reported here corroborate and extend the results of earlier experiments with fluorescein-labeled, size-fractionated dextran: diffusion of nonbinding particles in cytoplasm is hindered in a size-dependent manner. Extrapolation of the data suggests that particles larger than 260 A in radius may be completely nondiffusible in the cytoplasmic space. In contrast, diffusion of Ficoll in protein solutions of concentration comparable to the range reported for cytoplasm is not hindered in a size-dependent manner. Although we cannot at present distinguish among several physical chemical models for the organization of cytoplasm, these results make it clear that cytoplasm possesses some sort of higher-order intermolecular interactions (structure) not found in simple aqueous protein solutions, even at high concentration. These results also suggest that, for native cytoplasmic particles whose smallest radial dimension approaches 260 A, size may be as important a determinant of cytoplasmic diffusibility as binding specificity. This would include most endosomes, polyribosomes, and the larger multienzyme complexes.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

Biotechnology and Bioengineering, 2004

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.